InteractiveFly: GeneBrief

twine: Biological Overview | References

BIOLOGICAL OVERVIEW

The twine^HB5 mutation prevents spindle formation during the entry into meiosis in Drosophila males, but chromosome condensation and nuclear envelope breakdown both still occur. This suggests the possibility that this particular cdc25 phosphatase homologue is required to activate a p34cdc2 kinase required for only some of the events of this G2-M transition. In contrast, meiotic spindles do form in twine^HB5 females, although these appear abnormal. However, the female meiotic divisions do not arrest at metaphase I as in wild type, but continue repeatedly, leading to gross non-disjunction. Small chromatin masses, corresponding in size to the fourth chromosomes, often segregate properly to the spindle poles. These can persist into the embryos derived from twine^HB5 females, where they appear to participate in mitotic divisions on thin spindles. In addition, these embryos contain a small number of large chromatin masses that are not associated with spindles (White-Cooper, 1993).

cdc25 was first identified in fission yeast as a positive regulator of the 34 kDa mitotic kinase encoded by cdc2, a function that is opposed by the negative regulator wee1 (see Drosophila Wee and the zygotic cdc25 String). These proteins regulate the tyrosine 15 phosphorylation state, and thereby the activity, of p34cdc2, thus controlling the G2-M transition. wee1 encodes a protein kinase, which can phosphorylate tyrosine in vitro. Two homologues of cdc25 have been identified in Drosophila as the genes string and twine, both of which have been expressed in bacteria and shown to have tyrosine phosphatase activity and the capability to activate p34cdc2 in vitro. Moreover, both homologues are functional in fission yeast and will rescue a temperature-sensitive cdc25 fission yeast mutant. This formed the basis for the original isolation of twine (Jimenez, 1990), although the sequence conservation between all cdc25 genes enabled Courtot (1992) also to clone the twine gene using a PCR approach (White-Cooper, 1993).

Embryos homozygous for mutation in the cdc25 homologue string can complete the first 13 mitotic cycles that take place in the syncytial embryo utilising maternally provided string gene product, but fail to undertake the fourteenth round of mitosis that normally occurs following cellularisation. Unlike the earlier syncytial division cycles in which mitosis is synchronous, cycle 14 has an extended G2 period and mitoses occur in a series of spatially and temporally regulated domains. string transcription precedes these divisions by 25-35 minutes. If string is ectopically expressed in embryos under the control of the heat shock promoter, it will induce entry into mitosis throughout the embryo. Thus string seems to behave as the primary regulator of the G2-M transition in the newly cellularised embryo. string expression is also seen in dividing tissues in larval development. These expression patterns contrast with the distribution of twine transcripts, which, although present in the syncytial embryo as part of the maternal contribution, are otherwise not seen in somatic tissues throughout development (Alphey, 1992). string and twine expression show overlapping patterns during oogenesis, but are expressed in distinct regions of the testes (Alphey, 1992; Courtot, 1992). twine is expressed in the growing stage of primary spermatocytes in a manner that suggests a role in regulating the entry into meiosis, and analysis of a twine mutation has demonstrated a requirement for the gene not only in male, but also in female meiosis (Alphey, 1992; Courtot, 1992). This paper shows that twine is not required for all aspects of the entry into male meiosis, and mutation in twine leads to a variety of abnormal meiotic spindles and unusual chromosome segregation in female meiosis (White-Cooper, 1993).

Some aspects of the phenotypes resulting from a mutation in the Drosophila gene twine, one of two homologues of the fission yeast gene cdc25 have been described (Jimenez, 1990; Alphey, 1992). Previous studies suggested defects in the progression through meiosis in both males and females. In males, the meiotic divisions do not occur and so the cysts of primary spermatocytes remain with 16 cells, although they do undertake considerable further development, including elongation of the nuclei and formation of sperm-like structures. The synthesis of twine transcripts in the growing stage of primary spermatocytes suggests a role in regulating the entry into meiosis. This is analogous to the G2-M transition of the mitotic cycle in which the breakdown of the nuclear envelope, chromosome condensation, and the formation of the spindle are thought to be mediated through p34 cdc2 kinase, following its activation by dephosphorylation by the cdc25 phosphatase. The twine^HB5 allele was sequenced (Courtot, 1992) and shown to have a missense mutation that results in a conserved proline residue in the tyrosine phosphatase domain of the protein being changed to a leucine residue (White-Cooper, 1993).

The mutant phenotype of twine^HB5/Df is indistiguishable from that in homozygous twine^HB5 flies, indicating that twine^HB5 is an amorphic allele. This was also suggested by the failure of twine^HB5 to rescue a cdc25ts mutant of S. pombe (Courtot, 1991). It therefore seemed likely that p34cdc2 kinase regulation was not being correctly activated during meiosis in twine^HB5 mutants. It was therefore surprising to find that, contrary to expectations and to the report of Courtot (1992), chromosome condensation does occur in mutant twine^HB5 males and moreover it is accompanied by nuclear envelope breakdown. A spindle never forms, however, and so it seems that some aspects of the entry into meiosis can take place whereas others cannot (White-Cooper, 1993).

There are a number of possible explanations for these observations. Perhaps p34cdc2 does not mediate all aspects of the G2-M transition for the entry into male meiosis. This is not without precedent since in Aspergillus a kinase encoded by the gene nimA appears to mediate some events of the G2-M transition. Interestingly a gene for a related kinase, Nek1, has been identified that is expressed at high levels in meiotic germ cells in mouse. Alternatively some other 'cdk-like’ enzyme could mediate some of these steps. Another possibility is that twine and string independently activate different forms of p34cdc2 to mediate entry into meiosis, and thus in the twine mutant, the string-mediated steps can still occur. However, string transcripts are only seen at the apex of the testes in the progenitors of cells that will not undertake meiosis until about 90 hours later. The distribution of string protein in the testes is not known, but its seems unlikely that it would persist for this period of time, since in other systems cdc25 instability appears to be a key feature of its function as a mitotic regulator. An alternative is that some forms of the p34cdc2 complex might not be regulated by the phosphorylation/dephosphorylation of tyrosine 15. In extracts of activated Xenopus eggs p34cdc2 complexed to cyclin A is not subject to inhibitory phosphorylation of tyrosine 15, in contrast to the p34cdc2/cyclin B complex. If this were the case in the Drosophila spermatocyte, twine function would only be required to activate the p34cdc2/cyclin B complex. This would then be consistent with growing evidence in support of differing roles for the cyclin A- and cyclin B-associated p34cdc2 kinases in modifying microtubule behaviour. The cyclin B-associated enzyme is required to bring about the specific and abrupt shortening of interphase microtubules crucial in the establishment of the spindle, and in several organisms, including Drosophila, cyclin B has been demonstrated to associate with the polar regions of the spindle. The failure of the spindle to form in twine mutants could reflect a specific role in the activation of p34cdc2/cyclin B (White-Cooper, 1993).

In contrast to the meiotic block seen in twine^HB5 males, meiosis continues abnormally in females. Female meiosis normally arrests at metaphase I in stage 14 of oogenesis and remains blocked until the egg passes through the oviduct. The phenotype that observed in twine mutant females suggests that twine function is required to maintain this arrest by keeping p34cdc2 dephosphorylated at tyrosine 15 and thereby active. In Drosophila, meiotic recombination only occurs in the female. Thus it might be expected that the mechanisms regulating entry into the first meiotic division might differ between the sexes, since recombination requires the assembly of synaptonemal complexes and exchange nodules in what is essentially an extended prophase. The mechanism whereby the meiotic spindle is established in female meiosis is also quite characteristic, and probably explains the differing requirement for twine (and p34cdc2) function between male and female meiosis (White-Cooper, 1993).

A cytological study of spindle assembly in female meiosis led Theurkauf (1992) to propose that the major microtubule nucleating activity is provided by paired centromeres of the major chromosomes rather than the centrosomes. Such diverse mechanisms of spindle formation might be expected to be under different regulation, and so spindle formation in the female may not be blocked by the twine mutation, as it is in male meiosis. The bundling of microtubules emanating from the chromosomal nucleation points requires the activity of a kinesin-like molecule encoded by the ncd gene. In ncd mutants, this bundling is not complete, leading to spindles with broad poles that are often distorted around the metaphase plate, and which resemble the abnormal twine spindles. Normally an equilibrium exists at metaphase I in which the chromsomes that have undergone recombination remain at the equator still connected through chiasmata that will eventually ensure their correct segregation. The separation of nonexchange chromosomes is controlled in part through the kinesin-like protein encoded by no distributive disjunction (nod). This imparts a force upon these chromosomes in the direction of the metaphase plate, and is counteracted by a poleward directed force that allows non-exchange chromosomes to move toward the poles in a size-dependent manner. In this way, the tiny fourth chromosomes become positioned between the poles and the equator. This type of arrangement is not seen in the second meiotic metaphase in which all chromosomes align on the metaphase plate before undertaking the equational division. Premature separation of the fourth chromsome is seen in the multiple meiotic-like divisions that occur in twine oocytes. If these are repeated attempts at the reductional division, then this would explain the dramatic non-disjunction that occurs during twine meiosis. Mutation in nod leads to the dissociation of non-exchange chromosomes from the spindle or their premature movement to the pole. Similar events can also be seen in twine mutants (White-Cooper, 1993).

Arrest at metaphase I in female meiosis is normally also dependent upon recombination having taken place to produce chiasmate bivalents. Thus, in mutants that prevent recombination, the meiotic arrest at metaphase I does not occur. However, the absence of any significant zygotic lethality indicates that meiosis is otherwise normal and relies entirely upon the mechanisms for segregating non-exchange chromosomes. Thus the failure to arrest in twine mutants differs profoundly from the effects of mutations preventing meiotic recombination. It has been suggested that the formation of chiasmata leads to the establishment of mechanical tension at the metaphase plate that signals a meiotic block. The gross abnormalities observed in meiosis in twine females suggests that its function is likely to be a prerequisite for the block imposed through the mechanism that senses the presence of chiasmata (White-Cooper, 1993).

The Drosophila cdc25 homolog twine is required for meiosis

A second cdc25 homolog has been identified in Drosophila. In contrast to string (the first homolog identified in Drosophila) this second homolog, twine, does not function in the mitotic cell cycle, but is specialized for meiosis. Expression of twine was observed exclusively in male and female gonads. twine transcripts are present in germ cells during meiosis, and appear only late during gametogenesis, well after the end of the mitotic germ cell divisions. The sterile Drosophila mutant, mat(2)synHB5, which had previously been isolated and mapped to the same genomic region as twine (35F), was found to carry a missense mutation in the twine gene. This missense mutation in twine abolished its ability to complement a mutation in Schizosaccharomyces pombe cdc25. Phenotypic analysis of mat(2)synHB5 mutant flies revealed a complete block of meiosis in males and severe meiotic defects in females (Courtot, 1992. Full text of article).

Meiosis in males homozygous for mat(2)synHB5 is completely blocked. Meiotic figures with condensed chromosomes were never observed in mutant testes indicating that twine is required for entry into meiosis. Other aspects of spermatogenesis are not affected. The development and growth of premeiotic cysts appears completely normal. Moreover, postmeiotic differentiation processes also continue despite the absence of meiosis. Sperm tails elongate and the formation of sperm heads is attempted in mutant testes, although the compaction of the premeiotic, presumably 4N nuclei into the typical rod shape does not occur to the same extent as in the case of the postmeiotic 1N nuclei in wild-type testes (Courtot, 1992).

twine expression starts during the growth phase of cysts many hours before the onset of the meiotic divisions. string expression on the other hand is not observed during the meiotic stages. If this does not simply reflect an inability to detect transient, low level expression, it indicates that entry into meiosis is controlled by a mechanism different from that controlling entry into mitosis in the cellularized embryo, where the transcriptional control of string expression is thought to determine the time of entry into mitosis. During the embryonic cell division cycles, string expression is sufficient to force G2-cells into mitosis, and starts immediately (about 25 minutes) before mitosis in an intricate pattern which accurately anticipates the pattern of the subsequent division (Courtot, 1992).

As in testes from homozygous mat(2)synHB5 males, mitotic divisions of germ line cells were completely normal in mutant females and defects were only observed during meiosis. However, whereas entry into meiosis appears to be completely blocked in mutant males, it is still accomplished in mutant females. Nuclear envelope breakdown and chromosome condensation occur at the correct stage in mutant oocytes. But, in contrast to wild-type oocytes, mutant oocytes do not arrest at metaphase of the first meiotic division. Instead, chromosomes are replicated and dispersed into irregular nuclei of variable size. Such irregular nuclei were never observed in mutant testes. The extent of this chromatin dispersal in mature mutant oocytes varies considerably. This variability most likely reflects the fact that mature stage-14 oocytes can be retained for several hours before fertilization and egg deposition. It is assumed that the severity of the phenotype increases with increasing retention time, which is affected by feeding of the adult females. The observation that the proportion of eggs with few nuclei is higher in collections from well fed females than in collections from starved females is consistent with this assumption (Courtot, 1992).

Analysis of string expression suggests an attractive explanation for the phenotypic differences observed in males and females. Whereas no string transcripts were detected during the meiotic stages of spermatogenesis, string transcripts are clearly present in oocytes undergoing meiosis. Normal levels of string transcripts were also detected in mutant oocytes. It is therefore possible that the string activity allows entry into meiosis in mutant oocytes. The results of experiments in S. pombe clearly indicate that string and twine have similar activities since both can complement cdc25 alleles. Moreover, the presence of string activity in mutant oocytes might not only allow entry into meiosis but also further cell cycle progression (Courtot, 1992).

Current observations, however, showed that the oocyte nucleus does not proceed through regular mitotic cycles but is fragmented rapidly in the mutant oocytes. While an ordered cell cycle progression might be hampered for a variety of reasons, it is pointed out that the centrosome which might well be required for an ordered mitotic cycle is missing in late oocytes and is contributed only during fertilization by the sperm (Courtot, 1992).

According to this interpretation, twine would act formally analogous to CSF (cytostatic factor), an activity that causes the arrest at metaphase II during Xenopus egg maturation (see Hunt, 1992). Mechanistically, CSF acts by stabilizing the activity of MPF (maturation promoting factor), a factor which according to recent results represents the active complex of cyclin B and the p34cdc2 kinase. In Drosophila oocytes, MPF activity might be stabilized by twine activity and cause the arrest at metaphase of meiosis I until twine is inactivated after egg activation. Testing this idea will require a biochemical analysis of string and twine activity during female meiosis. Fortunately, recent progress in the development of methods allowing mass isolation and in vitro activation of mature oocytes should render such investigations feasible. In addition, such studies might also reveal which of the various cyclin-cdc2 (or cdc2-like) kinases is a target of twine activity (Courtot, 1992).

twine, a cdc25 homolog that functions in the male and female germline of Drosophila

twine is the second homolog of the fission yeast gene cdc25 to be found in Drosophila. Both string and twine cDNAs can rescue a temperature-sensitive cdc25 mutation in fission yeast, but not a deletion. The expression of string but not twine transcripts is detected in the proliferating cells of newly cellularized embryos, in third instar larval brains, and in imaginal discs. Both genes are abundantly expressed in nurse cells during oogenesis, the maternal transcripts persisting throughout the syncytial stage of embryonic development. In the testis, twine transcripts are seen in the growing stage of premeiotic cysts. Analysis of a twine mutant suggests a requirement for the gene during oogenesis, during syncytial embryonic development, and for male meiosis. Meiosis does not occur in homozygous twine males, which produce cysts containing 16 rather than 64 spermatids (Alphey, 1992).

Dmcdc2 kinase is required for both meiotic divisions during Drosophila spermatogenesis and is activated by the Twine/cdc25 phosphatase

The requirement for Drosophila cdc2 kinase during spermatogenesis was analyzed after generating temperature-sensitive mutant lines (Dmcdc2ts) by re-constructing mutations known to result in temperature sensitivity in fission yeast cdc2+. While meiotic spindles and metaphase plates were never formed in Dmcdc2ts mutants at high temperature, chromosomes still condensed in late spermatocytes and spermatid differentiation (sperm head and tail formation) continued. The same phenotype was also observed in twine and twine, Dmcdc2ts double mutant testes, consistent with the idea that the cdc2 kinase activity required for meiotic divisions is activated by the Twine/cdc25 phosphatase. Confirming this notion, it was found that ectopic expression of the String/cdc25 phosphatase, which is known to activate the cdc2 kinase before mitosis, results in a partial rescue of meiotic divisions in twine mutant testis (Sigrist, 1995).

Zygotic degradation of two maternal Cdc25 mRNAs terminates Drosophila's early cell cycle program

In Drosophila embryos the maternal/zygotic transition (MZT) in cell cycle control normally follows mitosis 13. This study shows that this transition requires degradation of two maternal mRNAs, string and twine, which encode Cdc25 phosphatases. Although twine is essential for meiosis and string is essential for most mitotic cycles, the two genes have mutually complementing, overlapping functions in the female germ line and the early embryo. Deletion of both gene products from the female germ line arrests germ-line development. Reducing the maternal dose of both products can lower the number of early embryonic mitoses to 12, whereas increasing maternal Cdc25_twine can increase the number of early mitoses to 14. Blocking the activation of zygotic transcription stabilizes maternal string and twine mRNAs and also allows an extra maternal mitosis, which is Cdc25 dependent. It is proposed that Drosophila's MZT comprises a chain reaction in which (1) proliferating nuclei deplete factors (probably mitotic cyclins) required for cell cycle progression; (2) this depletion causes the elongation of interphases and allows zygotic transcription; (3) new gene products accumulate that promote degradation of maternal mRNAs, including string and twine; and (4) consequent loss of Cdc25 phosphatase activity allows inhibitory phosphorylation of Cdc2 by Wee kinase, effecting G2 arrest. Unlike timing or counting mechanisms, this mechanism can compensate for losses or additions of nuclei by altering the timing and number of the maternal cycles and thus will always generate the correct cell density at the MZT (Edgar, 1996).

Wild-type Drosophila embryos always have 13 rapid, synchronous, maternally driven mitoses before they arrest in their first G2 period, cellularize, and assume zygotic control the cell cycle. The present work shows that embryos with increased maternal supplies of Cdc25 can have 14 maternally driven mitoses, whereas embryos with reduced maternal Cdc25 often have only 12. Thus maternal Cdc25 phosphatases are dosage-sensitive regulators that can determine how many mitoses occur before the transition to zygotic cell cycle control (Edgar, 1996).

It was also shown that blocking zygotic transcriptional activation by an early injection of α-amanitin stabilizes maternal Cdc25 mRNAs and, like raising Cdc25 dosage, allows an extra maternally driven cell cycle. Although maternal Cdc25 String protein is degraded normally after α-amanitin injection, dosage experiments suggest that Cdc25 String and Cdc25 Twine are nevertheless required for the α-amanitin-induced division cycle. These apparently paradoxical results may be reconciled by the likelihood that new Cdc25 String and/or Cdc25 Twine proteins are translated from the maternal mRNAs that are stabilized after α-amanitin injection, and that these accumulate to a threshold sufficient to drive the extra division. This interpretation is consistent with the finding that the stability of Cdc25 String protein is cell-cycle regulated, and with the detection of Cdc2 that is not Y15 phosphorylated {and is presumably active) during the α-amanitin-induced division. The simplest conclusion to be drawn from these findings is that degradation of maternal Cdc25 mRNAs is a critical event in the maternal/zygotic transition (MZT), and that this degradation is timed by the activation of zygotic transcription (Edgar, 1996).

By drawing the present work together with earlier observations a general model can be constructed that explains many aspects of the MZT. The maternal cell cycle oscillator appears to be inactivated as the final event in a chain reaction that starts at fertilization. Proliferation of the embryonic nuclei is the initial driving force of this reaction: As these nuclei multiply they progressively deplete something required for cell cycle progression, and this causes lengthening of interphases starting in cycle 10. Dosage experiments suggest that the first critical factors to be titrated out are probably mitotic Cyclins A and B, and not Cdc25 phosphatases. The amount of cyclin degraded at mitosis is proportional to the number of nuclei dividing in the embryonic cytoplasm, making cyclin depletion closely linked to nuclear proliferation. Beginning at mitosis 10 cyclins appear to be degraded to below the threshold required for mitosis, and an interphase lag occurs during which cyclins must reaccumulate (Edgar, 1996).

The second event in the chain reaction leading to the MZT is the activation of zygotic transcription. Activation of most genes appears to require slowing of the cell cycle in both Drosophila and Xenopus embryos. This may be because transcription is mechanically suppressed by DNA replication and chromosome condensation, because transcriptional repressors are titrated out of the embryonic cytoplasm by the proliferating nuclei, or because active Cdc2 kinase represses transcription by phosphorylating components of the transcription apparatus. This last explanation is particularly attractive because transient inactivations of Cdc2 kinase are first detected around cycle 10, just as general transcriptional activation begins. Regardless of which mechanism initiates transcription the result is the same: New gene products accumulate that promote degradation of maternal mRNAs including String and Twine. This causes Cdc25 phosphatase activity to plummet and allows inhibitory phosphorylation of Cdc2 by the Dweel kinase, a constitutively expressed maternal product. The result is cell cycle arrest in G2-14 (Edgar, 1996).

Although this proposed mechanism for the MZT is consistent with virtually all previous studies of the early Drosophila embryo, it should be noted that it is inconsistent with a recent report from Myers (1995). That study tracked string mRNA stability after α-amanitin treatment of early embryos but, in contrast to the current results, found that string mRNA was not stabilized. This inconsistency is ascribed to the possibility that the method used for delivering α-amanitin -- embryo permeabilization rather than injection -- did not inhibit transcription rapidly or completely enough to block the activation of the RNA degradation. In the current experiments it was noticed that embryos injected with α-amanitin later than cycle 6 often fail to have an extra division and have incomplete stabilization of maternal Cdc25 mRNAs. This suggests that even small amounts of transcription are sufficient to trigger turnover of maternal mRNAs (Edgar, 1996).

The mechanism that is proposed for Drosophila's MZT has several properties that are advantageous for the embryo. First, the functional coupling of several reactions that are progressive (cyclin depletion, cycle slowdown, transcriptional activation, and RNA turnover) makes for a switch that will inactivate the maternal oscillator rapidly and discreetly within one cell cycle. The coincident acceleration of RNA turnover, elongation of S phases, and the acquisition of an S/M checkpoint creates a time window in cycle 14 (45 minutes) that is ample for degradation of Cdc25 mRNAs, even when their levels are abnormally high. In contrast, the corresponding time window in cycle 13 is much shorter (<18 minutes) and occurs when RNA turnover is much slower, making premature degradation of Cdc25 mRNAs improbable even when their levels are abnormally low. Thus the switch is relatively resistant to environmental or genetic variations that might alter the activity of its components (Edgar, 1996).

As well as constituting a robust switch, the proposed coupled reaction mechanism may explain how Drosophila embryos add early cycles to compensate for lost or defective (nondividing) nuclei. Coupling the activation of RNA turnover to nuclear proliferation allows Cdc25 mRNA degradation to be delayed when nuclei are lost in the early cycles, and thus ensures that the MZT will occur at the correct cell density even if more cycles are required to achieve this density. When compared to proposed mechanisms that count the number of cell cycles or the time elapsed from fertilization, this type of switch would seem to have a great selective advantage (Edgar, 1996).

Transcriptional and post-transcriptional control mechanisms coordinate the onset of spermatid differentiation with meiosis I in Drosophila

always early (aly) is required for cyclin B and twine expression in primary spermatocytes. The level of cyclin B protein is reduced significantly in aly mutant testes compared to wild type. Although low levels are still detected by Western blotting of testis extracts, the three different aly alleles tested all cause a similar marked reduction in cyclin B protein level when compared to wild-type testis extracts. Cyclin B protein appears to be expressed at normal levels in can, mia and sa mutant testes, indicating that these genes might influence cell cycle progression through a biochemically distinct pathway from aly. aly is not required for the expression of all cell cycle genes, since both Cyclin A and Cdc2 proteins are expressed at high levels in aly testes (White-Cooper, 1998).

The wild-type function of aly is required for the transcription or accumulation of both cyclin B and twine mRNAs. In wild-type testes, cyclin B transcripts are detected by in situ hybridization at low levels in the mitotic cells at the apical tip, but are not detected at the position where cells undergo pre-meiotic S-phase. Cyclin B message is abundant throughout the primary spermatocyte stage, accumulating to very high levels in mature primary spermatocytes. Cyclin B mRNA is detected in meiotically dividing cells, but is absent from the post-meiotic stages. In aly mutant testes cyclin B transcript is detected in the mitotic cells at a level comparable to wild type. However cyclin B mRNA is not detectable in aly mutant primary spermatocytes. The expression pattern of twine mRNA in wild-type testes is similar to that of cyclin B, except that twine mRNA is not detected in the mitotic cells at the apical tip of the testis. In aly mutant testes twine mRNA is not detected by in situ hybridization. can, mia and sa mutant testes express both twine and cyclin B mRNA at normal levels (White-Cooper, 1998).

The lack of cyclin B and twine mRNAs in aly mutant spermatocytes is not due to a general defect in transcription, since cyclin A and other messages are abundant in the mutant spermatocytes. In wild-type testes cyclin A mRNA is detected at low levels in both mitotic and S-phase cells at the apical tip of the testis. Cyclin A shows high levels of expression throughout the primary spermatocyte stage, with the message disappearing during meiosis. In aly, can, mia and sa mutant testes cyclin A mRNA is expressed in mitotic and S-phase cells and spermatocytes as in wild type. However cyclin A transcript levels remain high in the arrested mature primary spermatocytes, only disappearing at the base of the testes where the cells finally degenerate, suggesting that the wild-type function of aly, can, mia and sa is required directly or indirectly for the normal shut down of transcription and/or turnover of cyclin A message at meiosis (White-Cooper, 1998).

The timing of entry into the meiotic divisions in wild type may be controlled by post-transcriptionally regulated accumulation of Cyclin B and Twine protein. Although Cyclin B mRNA is expressed at high levels in early spermatocytes, the accumulation of Cyclin B protein is delayed in wild-type testis until the late primary spermatocyte stage. Cyclin B protein begins to accumulate in the cytoplasm of late primary spermatocytes as chromosome condensation is initiated just before the entry into the first meiotic division and is present at high levels in pro-metaphase I cells. Cyclin B protein is degraded at the metaphase to anaphase transition of meiosis I, and reaccumulates in preparation for the second meiotic division. In aly mutant testis Cyclin B protein is detected in the mitotic cells at the apical tip, but does not accumulate in the mutant spermatocytes. Twine protein is likewise delayed until just before the entry into the first meiotic division, days after the transcript is first detected. Neither protein nor mRNA is detected in an aly mutant background (White-Cooper, 1998).

aly, can, mia and sa are required for accumulation of Twine protein but not twine transcript in late primary spermatocytes. These three meiotic arrest genes are required for the expression of fuzzy onions, whose product is required for mitochondrial fusion in early spermatids. Similarly, severe reductions in message level are observed for Male-specific RNA 87F (Mst87F), a gene normally transcribed in primary spermatocytes but not translated until mid- to late-spermatid stages, days after the completion of meiosis. gonadal, which is expressed as two differentially terminated variants in the testis, shows dramatic reduction of both variants in can, mia and sa mutant testis. It is proposed that the can, mia and sa gene products act together or in a pathway to turn on transcription of spermatid differentiation genes, and that aly acts upstream of can, mia and sa to regulate spermatid differentiation. It is also proposed that control of translation or protein stability regulates entry into the first meiotic division. It is suggested that a gene or genes transcribed under the control of can, mia and sa allow(s) accumulation of Twine protein, thus coordinating meiotic division with onset of spermatid differentiation (White-Cooper, 1998).

aly, can, mia and sa are required for the transcription in primary spermatocytes of several genes involved in postmeiotic spermatid differentiation. The fuzzy onions (fzo) gene product is required for mitochondrial fusion in early haploid spermatids. fzo transcription initiates in early primary spermatocytes and the mRNA is present throughout the growing stages in wild type. fzo mRNA is greatly reduced in aly, can, mia and sa testes, despite the presence of primary spermatocytes in the mutant tissue. Message levels in mutant testes ranged from undetectable to low levels under conditions in which the in situ hybridization signal in wild type was strong, indicating that transcription may be reduced to a low basal level, but not entirely turned off. Similarly severe reductions in message level were observed for Mst87F, a gene normally transcribed in primary spermatocytes but not translated until mid- to late-spermatid stages, days after the completion of meiosis. Several other genes also showed dramatic reductions in transcript levels in meiotic arrest mutant testes when assayed by in situ hybridization. Reduced transcript levels in aly, can, mia and sa spermatocytes are not due to a general defect in transcription since a number of genes were transcribed at normal levels in mutant spermatocytes (White-Cooper, 1998).

Comparison of the effects of aly, can, mia and sa mutations on transcript levels suggests that genes normally transcribed in primary spermatocytes can be grouped into three classes. The transcription of the first (general) class of genes is independent of aly, can, mia and sa function. The second (meiotic) class of genes requires the normal function of aly, but not can, mia or sa. Expression of the third (spermiogenic) class of genes requires the wild-type activity of all four of the meiotic arrest genes (White-Cooper, 1998).

Although mutations in aly, can, mia and sa appear to cause arrest at the same point in the G2-M transition of meiosis I (Lin, 1996), the genes apparently control cell cycle progression by different biochemical mechanisms. aly, but not can, mia or sa, is required for the transcription of cyclin B and twine. The wild-type function of can, mia and sa instead appears to be required either to allow translation of twine message or to stabilize twine protein in mature primary spermatocytes. In either case aly, can, mia or sa mutations presumably cause cell cycle arrest at the same point in the G2-M transition, due to lack of active Cdc2/Cyclin B kinase complex. Cdc2 protein resolves into two distinct isoforms in Western blots. The slower migrating form, which is enriched compared to the faster migrating form in twine mutant testes, has been identified as a hyperphosphorylated, inactive form. The slower migrating form of Cdc2 also appears to be enriched compared to the faster migrating form in aly, can and sa. Production of Twine protein, but not Cyclin B, is dependent on can, mia and sa. Thus, although both Cyclin B and Twine protein accumulation are regulated posttranscriptionally in wild-type testes, the genetic control of their expression is different (White-Cooper, 1998).

It is proposed that can, mia, and sa act together or in a pathway to activate a tissue and stage-specific transcription program in primary spermatocytes, and that failure to initiate this program results in a global block in spermatid differentiation due to the lack of an array of gene products. The wild-type functions of can, mia and sa appear to be required for transcription in primary spermatocytes of a set of genes encoding products involved in post-meiotic spermatid differentiation. Transcription of these genes is initiated early in the primary spermatocyte stage, several days before the arrest point of the meiotic arrest mutants. Therefore the lack of transcription of this set of genes is likely to be a cause of the arrest rather than merely a downstream consequence (White-Cooper, 1998).

Of the eight genes identified so far that depend on can, mia and sa for transcription, some information about the function or time of action of the gene products is available for six. The product of the fzo gene is required for mitochondrial fusion, a post-meiotic event. Although fzo is transcribed in primary spermatocytes, the protein is not detected by immunofluorescence staining of testes until late in meiosis II. Expression of Mst87F, of four related genes at 84D and two related genes at 98C is regulated translationally. Although mRNAs are transcribed in primary spermatocytes, the proteins do not accumulate until days after the meiotic divisions. All of these genes encode proteins that are components of a structure in the sperm tail. Similarly the translation of janB and dj mRNAs is delayed until several days after the completion of meiosis. While the function of LanB is unknown, Dj is thought to serve a dual function; it is found in the sperm tail, but sequence comparisons suggest a possible role as a chromatin component (White-Cooper, 1998).

It is proposed that aly acts upstream of can, mia and sa, possibly to control expression or activation of components of the transcription machinery that drives expression of the spermatid differentiation genes. Wild-type function of aly is required for accumulation of at least three different mRNAs in primary spermatocytes that are not dependent on can, mia and sa, suggesting that aly is able to act independently of can, mia and sa. However aly mutations cause the same phenotype, and fail to express the same set of spermatid differentiation genes, as can, mia and sa mutations. This strongly suggests that aly might affect spermatid differentiation through an effect on expression or activity of either can, mia or sa. The block in meiotic cell cycle progression in can, mia and sa mutant testes could be due to a cross-regulatory mechanism that serves to coordinate meiosis and the spermatid differentiation program. It is proposed that a gene or genes transcribed in primary spermatocytes under the control of can, mia and sa encode(s) product(s) required either directly or indirectly to relieve the translational repression of twine message or to stabilise the Twine protein. Such a cross-regulatory mechanism between the pathways leading to spermatid differentiation and meiosis could serve in wild type to ensure that spermatocytes do not enter meiotic division until the proposed transcription program for post-meiotic spermatid differentiation genes has been successfully initiated. A late cross-regulatory mechanism may also explain why mutations that block spermatid differentiation but not meiotic cell cycle progression have not yet been isolated (White-Cooper, 1998).

The signal that activates the G2/M transition in male meiosis could be accumulation of the product of the proposed crossregulatory gene to a threshold sufficient to allow expression of twine protein. Alternatively, timing of the G2/M transition for meiosis I could be set via a less direct mechanism, involving the proposed cross regulatory gene, but not set directly by its level. For example accumulation of Twine protein may require an extrinsic signal received or transduced by a gene or genes controlled by the can, mia and sa transcription program. The degenerative spermatocyte (des) gene, encoding a novel protein that may be membrane associated, is a possible candidate for a component of such a signalling pathway (White-Cooper, 1998).

Mutations in des, like aly, can, mia and sa, cause a block in both meiotic cell cycle progression and the onset of spermatid differentiation. des mutations are also semi-lethal, suggesting a role for this gene outside the testis. Pole cell transplantation experiments also implicate extracellular signals in the regulation of meiotic progression and spermatid differentiation. Male (XY) germ cells transplanted into a female (XX) host initiate spermatogenesis in the host ovary. However the transplanted cells arrest as primary spermatocytes and fail to undergo the meiotic divisions or initiate spermatid differentiation. Part of the program of spermatid differentiation regulated by can, mia and sa could act to destabilize or turn off transcription of certain messages expressed in primary spermatocytes but not needed or deleterious after meiosis. In wild-type testes, cyclin A mRNA is present in primary spermatocytes but not detectable in post-meiotic cells. Loss of cyclin A mRNA could be an important mechanism to prevent DNA replication during meiosis II or in haploid spermatids. In wild-type testes Cyclin A protein is degraded at metaphase I and is not resynthesised for the second meiotic division. In males mutant for aly, can, mia or sa, cyclin A message and Cyclin A protein (Lin, 1996) persist in the arrested primary spermatocytes, suggesting that the wild-type function of the meiotic arrest genes and/or the transcription program they control is required directly or indirectly for disappearance of cyclin A message midway through spermatogenesis. A similar effect on message stability was seen for all of the other pre-meiotic genes tested (White-Cooper, 1998).

Yeast meiosis bears striking similarities to Drosophila spermatogenesis. In both cases S phase is followed by an extended G2 phase, characterized by high levels of transcription of genes required for meiosis and subsequent differentiation into spores or sperm. Many yeast mutants, including certain alleles of cdc2 in S. pombe, are analogous to twine, in that the mutant cells fail to complete one or both of the meiotic divisions, but still differentiate into spores. However meiosis and differentiation are coordinated, since mutations in some genes, mei4 in S. pombe or NDT80 in S. cerevisiae, like the meiotic arrest mutants of Drosophila, block both the meiotic division cycle and subsequent differentiation. The failure to accumulate both cell cycle and spermiogenesis mRNAs in aly mutants suggests that there may be parallels in the genetic control of animal spermatogenesis and yeast sporulation (White-Cooper, 1998 amd references therein).

Post-transcriptional regulation of the meiotic Cdc25 protein Twine by the Dazl orthologue Boule

Boule is required for Twine protein expression. By assaying the progress of spermatogenesis in the twine partial loss of function background, boule is a candidate in vivo regulator of twine. Spermatocytes from males with partial loss of function for twine and with only a single wild-type copy of boule fail to enter meiosis. Moreover, these cells do not carry out the G2/M transition; unlike wild-type or twine mutant spermatocytes, these double mutant cells fail either to relocalize cyclin A from the cytoplasm to the nucleus or to form bipolar spindles (Maines, 1999).

To explore the mechanism by which boule acts as an enhancer of twe mutants, Twine expression was examined in genetic backgrounds deficient for boule, using a reporter construct. Expression of the Twine reporter protein is dramatically reduced in a boule mutant compared with wild type. This reduction occurs at the level of protein, and not RNA, accumulation, since the amounts of twine–lacZ and endogenous twine RNA present are not reduced but are instead increased in the absence of boule. Given that, in wild-type flies, Twine RNA accumulates well before the onset of meiosis, these data indicate that (1) Twine is translationally regulated and (2) efficient Twine translation requires Boule (Maines, 1999).

Support for this hypothesis is provided by a study of the patterns of Boule localization and Twine expression in developing spermatocytes. Boule protein translocates from the nucleus to the cytoplasm just before the first meiotic division, and is required in the cytoplasm for meiotic entry (Cheng, 1998). Boule relocalization is coincident with the first detectable expression of the Twine reporter, consistent with cytoplasmic Boule being essential in the translation of Twine protein (Maines, 1999).

Heterologous Twine expression rescues the boule meiotic-entry defect. If boule mutants fail in meiotic entry because of inadequate accumulation of Twine protein, heterologous expression of Twine should drive meiotic entry in a boule mutant. To test this prediction, Twine was expressed in a boule-independent manner by placing twine under the control of the spermatocyte-specific beta2-tubulin gene promoter and the 5' and 3' untranslated sequences of the beta2-tubulin gene. Expression of twine in this context is sufficient to restore fertility to twine mutant males (Maines, 1999).

Introduction of the beta2-twine construct into a boule mutant drives meiotic entry, with some cells completing at least one division in all individuals studied. Meiotic spindles, which are absent in boule mutant males lacking the beta2-twine transgene, are easily apparent in transformed lines. Although the observed rescue could result from Twine mRNA overexpression, this seems unlikely for two reasons: (1) amounts of endogenous Twine mRNA are already quite high in boule mutants; (2) the rescue is not dose dependent and the presence of either one or two copies of the -beta2-twine transgene has no deleterious effect in the wild type. Furthermore, the activity of the beta2-twine construct in a boule mutant does not reflect a general ability to drive meiotic entry. For example, although the male sterile phenotype produced by mutation of the pelota gene closely resembles that produced in twine or boule mutants, the beta2-twine construct does not restore meiotic entry in a pelota male. Thus, the beta2-twine transgene appears to specifically compensate for the defect in endogenous Twine translation in a boule mutant, thereby restoring meiotic entry (Maines, 1999).

Boule cannot be required to stabilize the Twine mRNA, because the Twine message is abundant in a genetic background lacking boule activity. Instead, Boule is likely to play a part in Twine translation. Given that Boule contains an RNA-recognition motif (RRM), it is thought that Boule could influence Twine expression through direct binding to the Twine mRNA. Twine is not, however, the sole target of Boule activity, since boule mutants show defects in spermatid differentiation that are absent in twine mutant males (Eberhart, 1996). Taken together, these data indicate that Boule may be required for both meiosis and spermatid differentiation, and may have a role in coordinating these two events (Maines, 1999).

The boule and twine gene products act downstream of a second set of genes required for meiotic entry. The products of these genes, which include spermatocyte arrest (sa) and meiosis I arrest (mia), are also required for the expression of Twine protein but not of twine mRNA (White-Cooper, 1998). Mutations in these genes result in a failure to accumulate Boule protein. This inability of sa and mia mutants to express Boule and therefore Twine, presumably contributes to their failure to initiate meiotic divisions (Maines, 1999).

Although proteins of the Dazl family are required for fertility in several organisms, little is known about their biochemical function. Given the similarities in sequence and in expression patterns among Boule and other Dazl-family members, and also in the phenotypes induced by mutation of these proteins, it is suggested that a role in translation or translational control will be a general property of such proteins. Moreover, given that inactivation of the murine Dazl protein results in a proliferation defect, it is possible that transcripts encoding Cdc25 proteins will prove to be a general target for Daz-related proteins (Maines, 1999 and references therein).

A genetic link between morphogenesis and cell division during formation of the ventral furrow in Drosophila

Tribbles activity regulates cell cycle by directly and posttranscriptionally affecting String expression. During early embryonic development, string is transcribed in a spatial pattern controlled by the anterior-posterior and dorsoventral patterning systems. Expression of String mRNA in a given mitotic domain precedes mitosis by a few minutes. By analyzing the exception to this rule found in domain 10 on the ventral side at the embryo, the tribbles mode of regulation was uncovered. Although string is expressed in these cells, they do not divide until they are internalized. This delay depends on the activity of the tribbles gene named after the small, round, fictional organisms (from the television series "Star Trek") that proliferate uncontrollably when they contact water. The tribbles effect is restricted to the ventral furrow, even though TRBL mRNA is also present outside of this domain and the trbl mutation can be rescued by uniform exogeneous expression. This suggests that trbl activity is triggered by an input which is present only in the ventral furrow region. Tribbles acts by specifically inducing degradation of the CDC25 mitotic activators String and Twine via the proteosome pathway. By regulating CDC25, Tribbles serves to coordinate entry into mitosis with morphogenesis and cell fate determination. In embryos mutant for either snail or twist, no ventral furrow forms and cells are shifted to more lateral fates. String mRNA is not present in domain 10 and mitotic patterns in the ventral region of these mutant embryos are difficult to evaluate. String mRNA is restored to wild-type levels in the prospective domain 10 of snail mutants carrying three copies of wild-type twist. In such mutants, the ventral cells are the first ones to divide, indicating that snail is required for the function of the ventral inhibitor. One possibility would be that the persistence of trbl expression in the ventral region requires mesodermal determination and thus wild-type snail activity. However, snail mutants show a normal pattern of trbl expression and maintain trbl expression in the ventral domain. Similarly, in twist homozygous mutants and in embryos homozygous for deficiencies for frs the expression of trbl is not changed. Because snail embryos do not show a ventral mitotic inhibition, even though their trbl expression is normal, it is concluded that some aspect of mesodermal determination mediated by snail is required for Trbl activation (Großhans, 2000).

A novel eIF4G homolog, Off-schedule, couples translational control to meiosis and differentiation in Drosophila spermatocytes

During spermatogenesis, cells coordinate differentiation with the meiotic cell cycle to generate functional gametes. The gene off-schedule, now termed eukaryotic translation initiation factor 4G2 by FlyBase, was identified as being essential for this coordinated control. During the meiotic G₂ phase, Drosophila ofs mutant germ cells do not reach their proper size and fail to execute meiosis or significant differentiation. The accumulation of four cell cycle regulators -- Cyclin A, Boule, Twine and Roughex -- is altered in these mutants, indicating that ofs reveals a novel branch of the pathway controlling meiosis and differentiation. Ofs is homologous to eukaryotic translation initiation factor eIF4G. The level of ofs expression in spermatocytes is much higher than for the known eIF4G ortholog (known as eIF-4G or eIF4G), suggesting that Ofs substitutes for this protein. Consistent with this, assays for association with mRNA cap complexes, as well as RNA-interference and phenotypic-rescue experiments, demonstrate that Ofs has eIF4G activity. Based on these studies, it is speculated that spermatocytes monitor G₂ growth as one means to coordinate the initiation of meiotic division and differentiation (Franklin-Dumont, 2007). A second study, co-published with the Franklin-Dumont paper, see Baker (2007) below, has reported similar findings.

Initiation is the rate-limiting step in translation and is the most common target of translational control. The mRNA 5' cap is bound by eIF4F, a heterotrimeric protein complex that is the focal point for initiation. eIF4G is the backbone of this complex; it interacts not only with eIF4E, but also with eIF4A, an RNA helicase that facilitates ribosome binding and its passage along the 5' untranslated region (UTR) towards the initiation codon. eIF4G also associates with eIF3, a multisubunit factor that bridges the proteins bound to the mRNA's 5' end with the 40S ribosomal subunit. This ribosomal subunit comes 'pre-charged' as a ternary complex composed of eIF2, GTP and the initiator methionine-transfer RNA. With the aid of eIF4 initiation factor as well as ATP, this agglomeration of RNA and protein is thought to scan the mRNA in the 5' to 3' direction. When it encounters an AUG start codon in an optimal context, other factors as well as the 60S ribosomal subunit are recruited and polypeptide chain elongation begins (Richter, 2005).

The eIF4E-eIF4G interface is an important target for translational control. The core portion of eIF4G that interacts with eIF4E is small -- about 15 amino-acid residues (Mader, 1995). Strikingly, several other proteins contain similar peptide motifs, and it is this region that competes with eIF4G for binding to eIF4E; in this manner they control the rate of 40S ribosomal subunit association with mRNA, and hence translation initiation. A clear demonstration of why the competition between eIF4G and other proteins for interaction with eIF4E is so effective in preventing translation comes from X-ray crystallographic analysis. Peptides derived from the regions of eIF4G and an eIF4E inhibitory protein called 4E-BP (for 4E-binding proteins, also known as PHAS-I for phosphorylated heat and acid soluble protein stimulated by insulin; see Drosophila Thor) form nearly identical α-helical structures that lie along the same convex region of eIF4E, some distance from this protein's cap binding site (Marcotrigiano, 1997; Matsuo, 1997). Peptides with the general sequence YXXXXLphi, where phi is any hydrophobic amino acid, would probably form similar α-helical structures, implying that other proteins containing this peptide motif could control translation initiation (Richter, 2005).

The original three eIF4E inhibitory proteins, the 4E-BPs, prevent eIF4F complex formation by sequestering available eIF4E. This sequestration results in the inhibition of translation of certain mRNAs that normally require high levels of available eIF4E (Gingras, 1999). eIF4E-binding proteins interact with the eIF4E on only specific mRNAs, and do so either because they also interact with certain RNA elements directly, or do so through affiliations with RNA binding proteins (Richter, 2005).

A novel eIF4G homolog, Off-schedule, couples translational control to meiosis and differentiation in Drosophila spermatocytes

In spermatogenesis, progenitor cells must execute the meiotic divisions in coordination with acquiring the specialized morphology and functionality of sperm. This conserved process is particularly amenable to analysis in Drosophila. The fly testis is a blind-ended tube organized as an assembly line for spermatogenesis. Germline stem cells at the blind end give rise to gonialblasts, which divide mitotically four times with incomplete cytokinesis to produce a cyst of 16 interconnected spermatogonia. These cells exit the mitotic cycle and enter meiosis as spermatocytes, exhibiting an extended G₂ phase characterized by a significant increase in cell mass and robust transcription. At the end of G₂, the spermatocytes undergo the meiotic divisions and begin the conversion from round spermatids to specialized spermatozoa (Franklin-Dumont, 2007).

Ten 'spermatocyte arrest' genes are required for both meiosis and differentiation and are sorted into two classes according to their molecular targets and specific role in promoting transcription. The always early (aly) class affects the transcription of meiotic genes such boule, twine and cyclin B, as well as that of differentiation genes such as fuzzy onions (fzo) and don juan. Notably, these mutations do not effect transcription of other spermatocyte genes, such as pelota, cyclin A and roughex. The Aly class proteins are thought to alter chromatin structure to permit the high levels of transcription necessary in spermatocytes. The cannonball (can) class affects boule and twine expression post-transcriptionally only and has no effect on cyclin B. The post-transcriptional effects must be indirect, because all can class loci encode testis-specific components of the general transcriptional machinery. Together, the spermatocyte arrest genes reveal how a diverse set of genes is selectively transcribed in spermatocytes (Franklin-Dumont, 2007).

The transcriptional regulatory pathway does not address the timing of meiotic entry and differentiation, however. Although transcripts necessary for these processes accumulate in early spermatocytes, the corresponding proteins do not appear until much later. Because there is little, if any, transcription after the G₂-M transition in flies, spermatocytes must delay meiotic division until all the necessary transcripts have accumulated. A similar dilemma exists during the mitotic cycle in yeast. For cells to maintain the same average size over several divisions, control points act during the gap phases and allow cell cycle progression only when the cell has reached a threshold size, with G₁ predominating in budding yeast and G₂ in fission yeast. Cell growth rates also feed back on mitotic cell cycle progression in Drosophila cells. Less is known about how growth might affect the specialized meiotic cell cycle (Franklin-Dumont, 2007).

Identification and characterization of off-schedule provides evidence that cell growth is linked to the coordination of meiosis and differentiation. Spermatocytes in ofs mutant males fail to execute the G₂-M transition of meiosis or substantive post-meiotic differentiation and have a significant cell size defect. The Off-schedule protein resembles the eukaryotic initiation factor 4G (eIF4G), which is a member of the eIF4F translation initiation complex and bridges mature mRNAs and the ribosome (Prevot, 2003). The eIF4G activity of Ofs is apparent in its ability to associate with mRNA caps and to functionally replace canonical eIF4G in cell culture. Because translation is primarily regulated at initiation, eIF4G is instrumental in determining the translational capacity of a cell and thus its ability to accumulate mass. Thus, the ofs mutant phenotype suggests that sufficient cell mass must accumulate before spermatocytes execute meiosis and differentiation (Franklin-Dumont, 2007).

Alignment among eIF4G sequences suggests that Ofs would be part of the eIF4F complex with eIF4A and eIF4E, and demonstration of its association with 7-methyl GTP Sepharose strongly supports this. Although binding of Ofs directly to eIF4A was not measured, alignment of human and fly eIF4G proteins shows conservation of three out of four sets of amino acids necessary to bind eIF4A (Imataka, 1997). Of 12 crucial residues, ten were identical in Ofs, one was a conservative (L>I) change, and the twelfth diverged in Drosophila eIF4G as well. With regard to eIF4E binding, the putative binding site in Ofs has an arginine substituted for the usual hydrophobic residue. However, a similar substitution is tolerated in Drosophila eIF4E binding protein 1 (Miron, 2001), and Baker (2007) presents evidence for interaction with Drosophila eIF4E1. Taken together, it is quite likely that Ofs participates in cap-dependent translation initiation (Franklin-Dumont, 2007).

eIF4G (CG10811) and Ofs (CG10192) appear to be the only two eIF4G proteins encoded in the fly genome. One other candidate, l(2)01424, is more related to the proposed translational inhibitor, NAT1/p97 (Rpn1)/DAP5, than to eIF4G proteins (Takahashi, 2005). Although the novel N-terminus of Ofs raised the possibility that it would play a role distinct from eIF4G, the data suggest that Ofs can act as the only eIF4G in cultured cells. Whether these two proteins always act redundantly in vivo cannot be assessed without mutations in eIF4G. Nevertheless, eIF4G, at its endogenous level, cannot substitute for Ofs in spermatocytes. Perhaps this is simply due to a relatively lower level of eIF4G compared with Ofs. Alternatively, Ofs might uniquely aid in the translation of a special class of mRNAs, specific to spermatocyte development. Perhaps sequences in its novel N-terminus assist in such a role. Although further experiments are needed to distinguish between these possibilities, one reason for a distinction between spermatocytes and other cells might be in their respective mode of growth control. In cultured eIF4G-deficient mitotic cells, the cell cycle effect observed was on G₁, whereas the defect in spermatocyte progression is in G₂. Although the G₁-S transition is the major control point for growth sensing in mitotic cells of the fly, G₂ might make more sense as the control point for meiosis, because it is during this phase of the cycle that spermatocytes need to prepare not just for division, but for differentiation. Furthermore, spermatocytes might commit to the meiotic cycle, versus returning to the mitotic cycle, during G₂, as is the case for the yeast Saccharomyces cerevisiae. Perhaps expressing a unique eIF4G (Ofs) in spermatocytes helps serve this role. Given the functional role for ofs, it is proposed that ofs henceforth be known as eIF4G2 (Franklin-Dumont, 2007).

Because ofs (eIF4G2) encodes the predominant eIF4G in spermatocytes, one might expect that mutant cells would exhibit decreased translation of many mRNAs. Just as a striking delay was found in Boule accumulation, other proteins would be expected to be similarly affected. Such a global deficit could account for the delayed development of these cells, and would be predicted to influence cell size, because the translational capacity of a cell predicts its ability to accumulate mass. Indeed, one of the earliest phenotypes in eIF4G2 spermatocytes was their small size. Yet, Aly accumulation appeared normal and Rux protein appeared to accumulate to an excess degree in early spermatocytes. These data demonstrate that some mRNAs are not affected by the translational deficit, and raise an alternative scenario wherein spermatocytes actively monitor their size. If they do not achieve proper growth, a checkpoint is induced to prohibit meiosis and differentiation. Because meiosis involves two cell divisions with little intervening interphase, size monitoring would be especially important before these cells commit to divide (Franklin-Dumont, 2007).

Circumstantial support for a growth checkpoint includes the accumulation of the Cdk inhibitor Rux, which leads to aberrant behavior of Cyclin A. In this model, the postulated checkpoint causes the striking delay in the accumulation of Boule, which, in turn, explains the delay in Twine accumulation. Eventually, Boule does accumulate to reasonable levels, perhaps as cells leak through the checkpoint, just as eventually occurs in mitotic checkpoints. However, by then, Cyclin A has been degraded, and without it, the eventual accumulation of Twine cannot trigger meiosis, so the checkpoint has succeeded (Franklin-Dumont, 2007).

To establish that a checkpoint exists, one would need to identify the sensor, which detects the problem, and effectors, which execute inhibitory functions until the cell resolves the problem. No candidate is available for the sensor that detects growth at this time, nor for effectors controlling differentiation. However, it can be speculated that Rux is one effector regulating the meiosis branch, where it could serve to inhibit Cyclin A-driven Cdc2 kinase activity (Avedisov, 2000). Rux is not the only effector regulating meiosis, however. Previous work showed that directly increasing the level of Rux only blocked entry into the second meiotic division (Gönczy, 1994). Consequently, the accumulation of Rux that is observed in eIF4G2 mutants cannot fully explain the absence of the first meiotic division or the defect in differentiation. As would be typical for cell cycle regulation, several effectors must be activated at once to completely block the G₂-M transition (Franklin-Dumont, 2007).

The existence of other effectors could explain why forcing early Twine accumulation failed to restore meiotic entry to eIF4G2 mutants in a rux background. Alternatively, there might be additional positive factors necessary for G₂-M transition that have not accumulated in eIF4G2 spermatocytes. Consistent with this, prior work driving expression of another Cdc25, string (stg), in early spermatocytes directed a normal rather than a precocious G₂-M transition. Thus, advancing Cdc25 activity is insufficient to trigger a precocious G₂-M even in the absence of a growth defect. Perhaps early spermatocytes have not had enough time to accumulate an essential component, such as Cyclin B, for the meiotic divisions. It was found that eIF4G2 mutant clones exhibit Cyclin B levels comparable to neighboring heterozygous cells. However, there is a peak in Cyclin B accumulation just prior to meiosis I, and Baker (2007) describes a deficit of this Cyclin B peak in eIF4G2 mutants. Thus, Cyclin B remains a candidate factor (Franklin-Dumont, 2007).

Whether a growth checkpoint exists or not, mass accumulation could be used to time the G₂-M transition by coupling rate-limiting cell cycle proteins to the translational capacity of the cell. In the budding yeast, S. cerevisiae, cyclin CLN3 (also known as YHC3) contains an upstream open reading frame in the 5' UTR that slows its translation in G₁ under poor growth conditions. Similarly, during G₂ in the fission yeast, Schizosaccharomyces pombe, accumulation of CDC25 is disproportionately affected by defects in translation. Perhaps the translation of Boule, along with a few other meiotic cell cycle regulators, is disproportionately affected when translation is compromised in spermatocytes. Although this should be investigated, this simpler model does not explain the aberrant accumulation of Rux and the nuclear sequestration of Cyclin A that was observed (Franklin-Dumont, 2007 and references therein).

The defects in differentiation in eIF4G2 mutants are not secondary to the meiotic block, because several cell cycle mutants fail to divide but still undergo substantial post-meiotic differentiation. Several spermatid differentiation genes, such as don juan and fuzzy onions, are transcribed in primary spermatocytes under the control of spermatocyte arrest genes. Translational control delays the accumulation of their protein products. This delay is functionally relevant, because precocious don juan accumulation leads to sterility. In principle, then, the lack of significant differentiation in eIF4G2 mutants could simply be due to a more pronounced translational delay for key differentiation genes. Alternatively, the block in differentiation might reflect a direct effect of the proposed growth checkpoint. Consistent with either model, the accumulation of the mitochondrial fusion protein Fuzzy onions is delayed, although this was not timed precisely. It is expected that other differentiation targets will also be abnormally delayed in eIF4G2 mutants (Franklin-Dumont, 2007).

There are striking parallels to the role of eIF4G2 during spermatogenesis in other organisms. For instance, there are also two major isoforms of eIF4G in Caenorhabditis elegans, encoded by ifg-1. When the longest isoform was depleted from the germ line, oocytes arrested in meiosis I (B. D. Keiper, personal communication to Franklin-Dumont, 2007). The requirement for ifg-1 in spermatogenesis has not yet been examined. However, one of the five isoforms of eIF4E in the worm, IFE-1, is clearly essential for spermatogenesis. RNA interference against ife-1 results in delayed meiotic progression, and in defective sperm, in both hermaphrodites and males (Amiri, 2001). Furthermore, mouse testes carrying the Y chromosome deletion Spy (also known as Eif2s3y-Mouse Genome Informatics) have a meiotic arrest phenotype due to a lack of EIF2 (also known as EIF2S2-Mouse Genome Informatics) function (Mazeyrat, 2001). Taken together, these examples suggest that translational control, and therefore possibly growth control, is a common theme for meiotic cycle cells (Franklin-Dumont, 2007).

Translational control of meiotic cell cycle progression and spermatid differentiation in male germ cells by a novel eIF4G homolog

Translational control is crucial for proper timing of developmental events that take place in the absence of transcription, as in meiotic activation in oocytes, early embryogenesis in many organisms, and spermatogenesis. Drosophila eIF4G2 is required specifically for male germ cells to undergo meiotic division and proper spermatid differentiation. Flies mutant for eIF4G2 are viable and female fertile but male sterile. Spermatocytes form, but the germ cells in mutant males skip the major events of the meiotic divisions and form aberrant spermatids with large nuclei. Consistent with the failure to undergo the meiotic divisions, function of eIF4G2 is required post-transcriptionally for normal accumulation of the core cell cycle regulatory proteins Twine and CycB in mature spermatocytes. Loss of eIF4G2 function also causes widespread defects in spermatid differentiation. Although differentiation markers Dj and Fzo are expressed in late-stage eIF4G2 mutant germ cells, several key steps of spermatid differentiation fail, including formation of a compact mitochondrial derivative and full elongation. These results suggest that an alternate form of the translation initiation machinery may be required for regulation and execution of key steps in male germ cell differentiation (Baker, 2007).

Although precedent for developmentally regulated translation initiation factor components comes from data on the cap binding protein eIF4E, such as Caenorhabditis elegans IFE-1 and IFE-4, and various eIF4Es from Drosophila, zebrafish and mammals, less is known about the potential for the core eIF4G subunit to show such tissue specificity. In a human hematopoetic stem cell line, eIF4GII is specifically recruited to 5' cap structures of mRNAs upon thrombopoietin-mediated induction of megakaryocyte differentiation, whereas levels of eIF4GI at the cap remain constant (Caron, 2004). However, this recruitment of eIF4GII could represent an overall increase in active initiation factor complex within differentiating megakaryocytes, rather than intrinsic transcript specificity on the part of eIF4GII (Baker, 2007).

Function of Drosophila eIF4G2 is required for both meiotic cell cycle progression and for many aspects of spermatid differentiation. However, loss of eIF4G2 does not cause meiotic arrest. The eIF4G2 loss-of-function phenotype in testes is different from the phenotype of mutations in the testis TAFs (tTAFs). In tTAF mutant males, spermatocytes arrest at the G2/M transition, fail to undergo meiotic division and show a complete absence of spermatid differentiation. By contrast, in eIF4G2 mutant males, germ cells appear to skip the major events of meiotic division but initiate spermatid differentiation. Germ cells in males mutant for the cell cycle phosphatase Twine, or cdc2^ts mutant males shifted to the non-permissive temperature, also skip the major events of meiotic division but proceed to execute spermatid differentiation. These data show that initiation and execution of the spermatid differentiation program can proceed even when male germ cells fail to execute the meiotic divisions (Baker, 2007).

The failure to undergo the meiotic divisions in eIF4G2 is likely to be due, at least in part, to failure to upregulate twine and cycB translation as spermatocytes mature. Although eIF4G2 is a homolog of a known translation initiation factor, and eIF4G2 mutant spermatocytes have defects in translation of cycB and twine, it is formally possible that eIF4G2 does not act directly on these transcripts, but rather on an upstream regulator of their translation. Future experiments will address whether eIF4G2 binds these two mRNAs, to determine whether its effect on their translation is likely to be direct or indirect (Baker, 2007).

Function of eIF4G2 also appears to be required for many aspects of spermatid differentiation. Although early spermatids form in eIF4G2 mutant males, the mitochondrial cloud fails to condense and form a compact mitochondrial derivative, and very little spermatid elongation takes place. The defects in spermatid differentiation in eIF4G2 mutant males are more severe than the defects observed in males mutant for the RNA-binding protein Boule, homolog of human BOULE and DAZL. These observations suggest that although both Boule and eIF4G2 are required for normal translation of twine, the requirement for eIF4G2 is more widespread. A broad requirement for eIF4G2 for timing or execution of many events during male germ cell differentiation is reflected in the pleiotropic nature of the eIF4G2 mutant phenotype in testes. Loss-of-function of eIF4G2 also affects spermatocyte growth as well as timing of events of the meiotic program in primary spermatocytes (Baker, 2007).

Given the broad defects observed in male germ cells, the predicted role of eIF4G2 in translation initiation, and the apparent reduction in transcript levels for the canonical eIF4G, it was surprising that Fzo and Dj proteins were expressed in spermatids from eIF4G2 mutant males. These findings suggest that eIF4G2 is not required (directly or indirectly) for translation of all mRNAs in mature spermatocytes and post-meiotic germ cells. It is possible that some of the canonical eIF4G protein persists from earlier germ cell stages, sufficient for translation of fzo and dj. However, if so, this is not sufficient for robust translation of cell cycle regulators twine and cycB in late spermatocytes, or for sufficient translation of additional mRNAs required for proper spermatid differentiation (Baker, 2007).

Search PubMed for articles about Drosophila Twine

Alphey, L., Jimenez, J., White-Cooper, H., Dawson, I., Nurse, P. and Glover, D. M. (1992). twine, a cdc25 homolog that functions in the male and female germline of Drosophila. Cell 69(6): 977-88. PubMed ID: 1606618

Amiri, A., Keiper, B. D., Kawasaki, I., Fan, Y., Kohara, Y., Rhoads, R. E. and Strome, S. (2001). An isoform of eIF4E is a component of germ granules and is required for spermatogenesis in C. elegans. Development 128: 3899-3912. PubMed ID: 11641215

Avedisov, S. N., Krasnoselskaya, I., Mortin, M. and Thomas, B. J. (2000). Roughex mediates G1 arrest through a physical association with Cyclin A. Mol. Cell. Biol. 20: 8220-8229. PubMed ID: 11027291

Baker, C. C. and Fuller, M. T. (2007). Translational control of meiotic cell cycle progression and spermatid differentiation in male germ cells by a novel eIF4G homolog. Development 134(15): 2863-9. PubMed ID: 17611220

Caron, S., Charon, M., Cramer, E., Sonenberg, N. and Dusanter-Fourt, I. (2004). Selective modification of eukaryotic initiation factor 4F (eIF4F) at the onset of cell differentiation: recruitment of eIF4GII and long-lasting phosphorylation of eIF4E. Mol. Cell. Biol. 24: 4920-4928. PubMed ID: 15143184

Courtot, C., Fankhauser, C., Simanis, V. and Lehner, C. F. (1992). The Drosophila cdc25 homolog twine is required for meiosis. Development 116(2): 405-16. PubMed ID: 1286615

Edgar, B. A. and Datar, S. A. (1996). Zygotic degradation of two maternal Cdc25 mRNAs terminates Drosophila's early cell cycle program. Genes Dev. 10(15): 1966-77. PubMed ID: 8756353

Franklin-Dumont, T. M., Chatterjee, C., Wasserman, S. A. and Dinardo, S. (2007). A novel eIF4G homolog, Off-schedule, couples translational control to meiosis and differentiation in Drosophila spermatocytes. Development 134(15): 2851-61. PubMed ID: 17611222

Gingras, A. C., Raught, B. and Sonenberg, N. (1999). eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. Annu. Rev. Biochem. 68: 913-963. PubMed ID: 10872469

Gonczy, P., Thomas, B. J. and DiNardo, S. (1994). roughex is a dose-dependent regulator of the second meiotic division during Drosophila spermatogenesis. Cell 77: 1015-1025. PubMed ID: 8020092

Großhans, J. and Wieschaus, E. (2000). A genetic link between morphogenesis and cell division during formation of the ventral furrow in Drosophila. Cell 101: 523-31. PubMed ID: 10850494

Hunt, T. (1992). Cell cycle arrest and c-mos. Nature 355: 587-588. PubMed ID: 1531697

Imataka, H. and Sonenberg, N. (1997). Human eukaryotic translation initiation factor 4G (eIF4G) possesses two separate and independent binding sites for eIF4A. Mol. Cell. Biol. 17: 6940-6947. PubMed ID: 9372926

Jimenez, J., Alphey, L., Nurse, P. and Glover, D. M. (1990). Complementation of fission yeast cdc2ts and cdc25ts mutants identifies two cell cycle genes from Drosophila: a cdc2 homologue and string. EMBO J. 9: 3565-3571. PubMed ID: 2120044

Mader, S., Lee, H., Pause, A. and Sonenberg, N. (1995). The translation initiation factor eIF-4E binds to a common motif shared by the translation factor eIF-4γ and the translational repressors 4E-binding proteins. Mol. Cell. Biol. 15: 4990-4997. PubMed ID: 7651417

Maines, J. Z. and Wasserman, S. A. (1999). Post-transcriptional regulation of the meiotic Cdc25 protein Twine by the Dazl orthologue Boule. Nat. Cell Biol. 1(3): 171-4. PubMed ID: 10559904

Marcotrigiano, J., Gingras, A. C., Sonenberg, N. and Burley, S. K. (1997). Cocrystal structure of the messenger RNA 5' cap-binding protein (eIF4E) bound to 7-methyl-GDP. Cell 89: 951-956. PubMed ID: 9200613

Matsuo, H. et al. (1997). Structure of translation factor eIF4E bound to m7GDP and interaction with 4E-binding protein. Nature Struct. Biol. 4: 717-724. PubMed ID: 9302999

Mazeyrat, S., Saut, N., Grigoriev, V., Mahadevaiah, S. K., Ojarikre, O. A., Rattigan, A., Bishop, C., Eicher, E. M., Mitchell, M. J. and Burgoyne, P. S. (2001). A Y-encoded subunit of the translation initiation factor Eif2 is essential for mouse spermatogenesis. Nat. Genet. 29: 49-53. PubMed ID: 11528390

Miron, M., et al. (2001). The translational inhibitor 4E-BP is an effector of PI(3)K/Akt signalling and cell growth in Drosophila. Nat. Cell Bio. 3: 596-601. PubMed ID: 11389445

Myers, F. A., Francis-Lang, H. and Newbury. S. F. (1995). Degradation of maternal string mRNA is controlled by proteins encoded on maternally contributed transcripts. Mech. Dev. 51(2-3): 217-26. PubMed ID: 7547469

Prevot, D., Darlix, J. L. and Ohlmann, T. (2003). Conducting the initiation of protein synthesis: the role of eIF4G. Biol. Cell 95: 141-156. PubMed ID: 12867079

Richter, J. D. and Sonenberg, N. (2005). Regulation of cap-dependent translation by eIF4E inhibitory proteins. Nature 433(7025): 477-80. PubMed ID: 15690031

Sigrist, S., Ried, G. and Lehner, C. F. (1995). Dmcdc2 kinase is required for both meiotic divisions during Drosophila spermatogenesis and is activated by the Twine/cdc25 phosphatase. Mech. Dev. 53(2): 247-60. PubMed ID: 8562426

Takahashi, K., Maruyama, M., Tokuzawa, Y., Murakami, M., Oda, Y., Yoshikane, N., Makabe, K. W., Ichisaka, T. and Yamanaka, S. (2005). Evolutionarily conserved non-AUG translation initiation in NAT1/p97/DAP5 (EIF4G2). Genomics 85: 360-371. PubMed ID: 15718103

Theurkauf, W. E. and Hawley, R. S. (1992). Meiotic spindle assembly in Drosophila females: behavior of nonexchange chromosomes and the effects of mutations in the nod kinesin-like protein. J. Cell Biol. 116(5): 1167-80. PubMed ID: 1740471

White-Cooper, H., Alphey, L. and Glover, D. M. (1993). The cdc25 homologue twine is required for only some aspects of the entry into meiosis in Drosophila. J. Cell Sci. 106: 1035-44. PubMed ID: 8126091

White-Cooper, H., Schafer, M. A., Alphey, L. S. and Fuller, M. T. (1998). Transcriptional and post-transcriptional control mechanisms coordinate the onset of spermatid differentiation with meiosis I in Drosophila. Development 125: 125-134. PubMed ID: 9389670

date revised: 25 July 2022

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